1,720,998 research outputs found
Targeting of aequorin chimeras to chloroplast subcompartments uncovers the generation of stimulus-specific organellar Ca2+ signals
No full textDissecting stimulus-specific Ca2+ signals in amyloplasts and chloroplasts of Arabidopsis thaliana cell suspension cultures
Get PDFCalcium is used by plants as an intracellular messenger in the detection of and response to a plethora of environmental stimuli and contributes to a fine-tuned internal regulation. Interest in the role of different subcellular compartments in Ca(2+) homeostasis and signalling has been growing in recent years. This work has evaluated the potential participation of non-green plastids and chloroplasts in the plant Ca(2+) signalling network using heterotrophic and autotrophic cell suspension cultures from Arabidopsis thaliana plant lines stably expressing the bioluminescent Ca(2+) reporter aequorin targeted to the plastid stroma. Our results indicate that both amyloplasts and chloroplasts are involved in transient Ca(2+) increases in the plastid stroma induced by several environmental stimuli, suggesting that these two functional types of plastids are endowed with similar mechanisms for handling Ca(2+). A comparison of the Ca(2+) trace kinetics recorded in parallel in the plastid stroma, the surface of the outer membrane of the plastid envelope, and the cytosol indicated that plastids play an essential role in switching off different cytosolic Ca(2+) signals. Interestingly, a transient stromal Ca(2+) signal in response to the light-to-dark transition was observed in chloroplasts, but not amyloplasts. Moreover, significant differences in the amplitude of specific plastidial Ca(2+) changes emerged when the photosynthetic metabolism of chloroplasts was reactivated by light. In summary, our work highlights differences between non-green plastids and chloroplasts in terms of Ca(2+) dynamics in response to environmental stimuli
The involvement of chloroplast Ca2+-permeable channels in plant organellar Ca2+ signalling
No full textThe role of calcium signaling in plants: from Arabidopsis to barley
Get PDFCa2+ is an important intracellular secondary messenger involved in many signal transduction pathways in plants. During abiotic and biotic stresses, specific Ca2+ signatures are formed within the plant cell causing a spike in the Ca2+ concentration that have a particular frequency, shape, and duration. Studying these Ca2+ signatures in detail will give a closer look into the plant's defense mechanisms caused by different gene regulations. Although, a lot of research on cytosolic and organellar Ca2+ transients has been performed in Arabidopsis thaliana in response to various stimuli, very little is known about the Ca2+ transients in crops such as Hordeum vulgare (barley). Therefore, the main aim of this project was to study the cytosolic Ca2+ transients caused in barley in response to oxidative and drought stress. For the first time, in this study, it was possible to target apoaequorin into the cytosol of barley to monitor the Ca2+ transients. The results showed stimulus specific Ca2+ signatures in barley and these signatures were compared with Arabidopsis. It was also revealed that these Ca2+ transients are tissue specific and dependent on the age of the barley seedling as well. By using inhibitors of the Ca2+ transients, it could be seen that the Ca2+ transients were caused by the influx of Ca2+ across the plasma membrane and from the Ca2+ release from internal stores like the endoplasmic reticulum. Furthermore, to investigate the gene expression involved in causing the Ca2+ transients in response to oxidative stress, RNA-sequencing was done using the leaf and root samples in the presence and absence of the Ca2+ channel inhibitor, lanthanum chloride. The results showed that there is a clear differentiation between stress responses that require a Ca2+ signal and those that are Ca2+ independent.
Ca2+ sensors, such as calmodulin (CaM) and CaM-like proteins (CML) transduce Ca2+ signals into a cellular response which are activated by different target proteins. In the second part of this work, a previously identified CaM/Ca2+ binding target protein called the Rieske iron-sulfur protein (RISP) was analyzed for its functionality and topology. To that end, the Ca2+-dependent CaM binding property of RISP was confirmed and the localization of the N- and C- terminus of the protein was found to be in the mitochondrial matrix indicating that the CaM/Ca2+ regulation occurs within the matrix. However, there still exists a possibility that the localization of the N-terminus of RISP is not exclusively fixed to the matrix but also to the inner membrane space
Analyse der amorphen und amyloiden Aggregatsequestrierung in Mitochondrien
Get PDFProteine müssen, um ihre Funktionen auszuführen, richtig gefaltet sein. Fehlgefaltete Proteine können Aggregate bilden, welche toxisch für die Zellen sind. Daher ist zum Schutz der Proteinhomöostase die Verhinderung der Aggregation zellulärer Polypeptide von wesentlicher Bedeutung. Dies ist jedoch nicht immer möglich und die Zelle muss derartige Aggregate detoxifizieren können. Durch die Ablagerung instabiler Proteine an bestimmten Sammelorten kann die Zelle sich schützen. Im Allgemeinen gibt es zwei Arten von Aggregaten: amorphe und amyloide. Für amorphe mitochondriale Aggregate wurde in Saccharomyces cerevisiae beobachtet, dass sie sich im intramitochondrial protein quality control compartment (IMiQ) ansammeln. Auf diese Weise werden die Mitochondrien und die Zelle geschützt. Der Effekt von amorpher Proteinaggregation auf die Gesamtheit aller Proteine war bis jetzt jedoch unklar. Daher wurden in dieser Studie zwei unterschiedliche quantitative Massenspektrometrie-Ansätze benutzt, um die Veränderungen im mitochondrialen Proteom und die Bindungsaffinität der mitochondrialen Proteine zu den Aggregaten zu analysieren. Die Proteinmengen des mitochondrialen Proteoms veränderten sich größtenteils nicht. Jedoch zeigten die meisten Proteine eine erhöhte Bindungsaffinität zu den Aggregaten. Besonders Proteine der Zellatmung und des Proteinqualitätskontrollsystems (PQC) waren an den Aggregaten angereichert. Im zweiten Teil dieser Arbeit wurde die Bildung von amyloiden Aggregaten in den Mitochondrien analysiert. Vergleichbar zum IMiQ sammelten sich amyloidogenen Proteine in wenigen Aggregaten im mitochondrialen Netzwerk an. Im Gegensatz zum IMiQ konnte diese Ansammlung die Zelle nur teilweise vor der von den Aggregaten ausgehenden Toxizität schützen. Sowohl für die amorphen als auch für die amyloiden Aggregate wurde eine Relokalisation der vorhandenen PQC-Proteine zu den Aggregaten hin beobachtet. Daher scheinen die Komponenten des PQC-Systems zu versuchen, die Aggregation zu verhindern und die löslichen Proteine von den instabilen Proteinen zu beschützen, in dem sie sich an den Aggregaten ansammeln. Dies ist jedoch nicht ausreichend für amyloide Aggregate, an denen sich über längere Zeit ein Großteil der PQC-Proteine ansammelte. So kann das PQC nicht seinen eigentlichen Funktionen nachgehen und die Zelle leidet. Daher bilden die amyloidogene Proteine ein toxischeres Aggregat als das IMiQ, welches nicht zu einer Detoxifizierung der instabilen Proteine führt.
Für die Bildung weniger Aggregate pro Zelle spielte die mitochondriale Fusion und Fission eine Rolle, unabhängig vom Aggregatstyp. Blockierung der mitochondrialen Fusion, wie in fzo1Δ, führte zu der Bildung von vielen kleinen Aggregaten im mitochondrialen Netzwerk. Blockierung der mitochondrialen Fission, wie in fis1Δ, führte hingegen zum Gegenteil, wenige große Aggregate. Der Wildtyp lag zwischen den beiden Extremen, aber es bildeten sich tendenziell eher wenige Aggregate. Die mitochondriale Dynamik ist ebenfalls involviert in der beobachteten asymmetrischen Vererbung der Aggregate. Im Wildtyp und in fis1Δ tendierten die Ablagerungsorte dazu sich am Nukleus zu bilden und so während der Zellteilung in der Mutterzelle zu bleiben. In fzo1Δ wurden die Aggregate nicht am Nukleus zurückgehalten und an die Tochterzelle vererbt. Somit ist eine funktionale mitochondriale Dynamik notwendig, um sicherzustellen, dass die Tochterzellen keine Aggregate erben. So können die Aggregate über die Zeit aus der Population verdünnt werden.Proteins must be correctly folded in order to fulfil their functions. Misfolded proteins can form aggregates that are toxic to the cells. Thus, the prevention of protein aggregation is essential to protect protein homeostasis. However, this is not always possible, and the cell needs a way to detoxify aggregates. This is achieved by sequestering the unstable proteins into distinct deposit sites. In general, there two are different types of aggregates: amorphous and amyloid. For amorphous mitochondrial aggregates it was observed in Saccharomyces cerevisiae that they accumulate in the intramitochondrial protein quality control compartment (IMiQ). Thereby, the mitochondria and the cell are protected. The effect of the IMiQ formation on all mitochondrial proteins so far was unclear. Thus, in this study, two quantitative mass spectrometry approaches were used to analyse the changes in the mitochondrial proteome and the binding affinity of the mitochondrial proteins to aggregates. The protein levels of the whole mitochondrial proteome remained largely unchanged while most proteins showed an increased binding affinity to the aggregates. Especially proteins of the cellular respiration and of the protein quality control system (PQC) were increased at the aggregates. In the second part of this work, the formation amyloid aggregates inside mitochondria were analysed. Similar to the IMiQ, the amyloidogenic proteins accumulated in a few distinct aggregates within the mitochondrial network. In contrast to the IMiQ, this accumulation could only partially protect the cells from toxicity exerted from the aggregates. For both the amorphous and the amyloid aggregates a re-localisation of the mitochondrial chaperones to the aggregates could be observed. Therefore, it seems that the already expressed proteins of the PQC system try to prevent aggregation and protect the soluble proteins from unstable proteins by accumulating at the aggregates. This is insufficient for the amyloid aggregates since there the majority of the PQC proteins accumulated over longer time periods. Thus, the PQC cannot carry out their actual functions and the cell suffers. Therefore, amyloidogenic proteins form a more toxic aggregate than the IMiQ, which does not lead to detoxification of the unstable proteins.
The mitochondrial fusion and fission played a role in the formation of a few aggregates per cell, independent of aggregate type. Blocking of the mitochondrial fission, as in fzo1Δ, led to many small aggregates in the mitochondrial network. In contrast, blocking of the mitochondrial fusion, as in fis1Δ, led to the opposite, few small aggregates. The wild type was between the two extremes but tended to form smaller aggregates. The mitochondrial dynamic is also involved in the observed asymmetric inheritance of the aggregates. In the wild type and in fis1Δ the deposition site tended to form at the nucleus and thus, was retained in the mother cell during cell division. In fzo1Δ the aggregates were not retained at the nucleus and were inherited to the daughter cell. Therefore, functional mitochondrial dynamics are necessary to ensure that daughter cells do not inherit aggregates and that the aggregates can be diluted from the population over time
The chloroplast calcium sensor protein CAS is part of the STN7/STN8 kinase phosphorylation network and is required for photoacclimation
Get PDFIn the present work, the phosphorylation profile of the chloroplast-localized Calcium Sensing Receptor (CAS) from A. thaliana was investigated and it could be shown that CAS is a target of multiple protein kinases acting differentially on several residues. Phosphoproteomics followed up by biochemical kinase assays strongly indicated that CAS is part of a phosphorylation network involving the important state transistion kinases STN7 and STN8 as well as, at least, one other calcium-regulated protein kinase. The role of light and calcium behind the activation of CAS phosphorylation was investigated in vitro in more detail and revealed that, at least under normal growth light conditions, STN7 might be the major kinase acting on CAS. The analysis was extended at the level of individual residues by studying the phylogenetic conservation of experimentally described phosphoresidues of the A. thaliana CAS isoform and by conducting in vitro assays using recombinant CAS fragments carrying mutations at selected positions. These analyses confirmed the relevance of the previously described phosphorylation site Thr-380, but also showed that other residues, in particular Thr-376, are possible targets. A spectrometric analysis of cas mutant plants revealed that CAS is very likely involved in the processes of photoacclimation to high light, as evidenced by a persistent strong excitation of PSI under this condition. The analysis of the phosphorylation status of several known thylakoid phosphoproteins in the cas mutant further suggested a potential defect in the dephosphorylation of the important light harvesting protein LHCII under high irradiance, suggesting a possible role for CAS in mediating the activity of the TAP38 phosphatase. In addition to the post-translational modification of CAS, a potential involvement of CAS in the circadian network of the chloroplast was explored. RT-PCR analyses revealed that the transcription of the CAS follows a regular diurnal rhythm, with the highest levels of its transcript levels found at the end of the night and lowest levels at the end of the day. Interestingly, the levels of the CAS protein appear to follow an opposite, 12 hours shifted rhythm, suggesting a physiological requirement for higher abundance of CAS during the day. In light of these results, a possible working model is discussed that integrates the evidences on the phosphorylation profiles and the diurnal regulation with a suggested involvement of CAS in photoacclimation responses
Calcium signaling in Arabidopsis and potato : From Ca<sup>2+</sup> transient to Ca<sup>2+</sup> (in)dependent protein regulation
No full textThis doctoral thesis presents an investigation into stress-related Ca2+ signaling in the model plant Arabidopsis thaliana and the crop Solanum tuberosum (potato). Chapter one describes the generation and characterization of Solanum tuberosum lines expressing the genetically encoded Ca2+ biosensor apoaequorin. We measured Ca2+ transients in response to different biotic and abiotic stimuli in comparison with the response observed in an established Arabidopsis apoaequorin line. We observed dose-dependent calcium signatures in response to a series of abiotic and biotic stress stimuli, including H2O2, NaCl, mannitol and the pathogen-associated molecular patterns (PAMPs), flg22 and Pep-13 with stimuli-specific kinetics. Direct comparison with Arabidopsis revealed differences in the kinetics and amplitude of Ca2+ transients between both species, implying species-specific sensitivity for different stress conditions. Furthermore, an additional potato sensor line expressing the redox-sensitive Grx1-roGFP2 probe was introduced. This system enabled the analysis of cytosolic redox dynamics in S. tuberosum and facilitated comparative studies with an existing redox-sensitive Arabidopsis sensor line. We observed that potato has a higher basal oxidative state compared to Arabidopsis, which may explain the differences in their Ca2+ signature in response to H2O2.
Chapter two shows a very different approach towards the elucidation of Ca2+ signaling. Here we performed a full proteome analysis, investigating the effect of Ca2+ transients induced by an oxidative stress stimulus (H2O2) on protein regulation after 10 and 30 minutes of stress treatment. To differentiate between Ca2+-dependent and Ca2+-independent protein responses to oxidative stress, a subset of samples was treated with the Ca2+ channel inhibitor LaCl3, thereby suppressing the transient Ca2+ signal. Comparative analysis of proteomic data between H2O2 and LaCl3 + H2O2 treated samples vs control samples provided insights into the distinct regulatory mechanisms associated with oxidative stress. We identified a high number of differentially abundant proteins (DAPs) after the combined treatment with LaCl3 + H2O2 that did not change upon treatment with H2O2 alone, indicating a strong attenuating effect of Ca2+ signaling on the oxidative stress response. In addition, we identified H2O2 responsive proteins after 10 and 30 minutes of stress treatment, resulting in two distinct subsets, indicating that the duration of the stress exposure significantly shapes proteome-wide adaptations. These H2O2 responsive proteins were further categorized as strictly Ca2+-dependent, partially Ca2+-dependent or Ca2+-independent. This categorisation revealed Ca2+-dependent shifts in three proteins between the two time points, suggesting a dynamic role of Ca2+ in regulating proteomic changes. Interestingly, strictly Ca2+-dependent proteins predominantly showed reduced abundance, implying a role for Ca2+ in protein degradation, while Ca2+-independent proteins generally exhibited increased abundance, suggesting potential upregulation, possibly through transcription.
Chapter three extends the findings of chapter two by employing a novel analytical framework, the Stress Knowledge Map (SKM), to further analyse the data. This computational tool compilates existing knowledge of plant stress response mechanisms extracted from published datasets, facilitating a systems-level analysis of stress signaling networks. The proteomic dataset obtained and analysed in Chapter 2 provided one of two case studies demonstration the application of SKM in complex analyses. It focussed on a subset of H2O2-responsive proteins and revealed that they could be linked to a source set of proteins related to Ca2+-signaling, either directly or through pathways of up to four steps. Integrating all identified shortest paths into a single network highlighted major network hubs connected to multiple known Ca2+ signaling genes, suggesting their potential role in regulating multiple targets
Molecular insights into abiotic stress responses in barley and Arabidopsis
Get PDFThis cumulative thesis, titled "Molecular insights into abiotic stress responses in barley and Arabidopsis", investigates two critical aspects of plant responses to abiotic stress. The first part explores hydrogen peroxide (H2O2) and its interaction with calcium (Ca2+) signalling in barley (Hordeum vulgare L.), while the second part examines drought stress regulation in Arabidopsis thaliana, revealing a novel mechanism involving the genes GASA3 and AFP1.
H2O2 plays a pivotal role in signalling pathways that enable plants to adapt to environmental challenges. Despite its importance, its transcriptomic impact remains underexplored. To address this, RNA-Seq analysis was used to examine changes in gene expression in barley roots and leaves after H2O2 treatment. This revealed 1883 differentially expressed genes (DEGs) in roots and 1001 in leaves, with most responses being tissue-specific. Only 209 DEGs were commonly regulated, and 37 showed opposing regulation across tissues. Gene ontology (GO) analysis highlighted the organ-specific nature of the response: leaf DEGs were enriched in hormone signalling, H2O2 response, and abiotic stress adaptation, while root DEGs were associated with H2O2 detoxification, glutathione metabolism, and cell wall modifications. A follow-up study examined the cross-talk between H2O2 and Ca2+ signalling using RNA-Seq under conditions that blocked Ca2+-transients. By comparing expression profiles from H2O2-only and LaCl3+H2O2 treatments, 331 Ca2+-dependent H2O2-responsive genes in leaves and 1320 in roots were identified and grouped into five and four clusters, respectively. A SKM network analysis further revealed transcription factors potentially governing this H2O2–Ca2+ cross-talk.
Drought is one of the most severe abiotic stresses impacting plant growth, development, and reproduction. Like H2O2, drought triggers extensive transcriptomic reprogramming. In this study, two strongly drought-induced genes, GASA3 and AFP1, were identified. Loss-of-function mutants showed enhanced drought resistance, while constitutive overexpression of either gene led to reduced tolerance. The gasa3afpl double mutant exhibited even greater resistance than single mutants. Both genes are also ABA-inducible, though GASA3 expression remained low in the absence of AFP1. The improved drought tolerance in mutants was linked to higher leaf water content due to smaller stomatal apertures and reduced transpiration. Additionally, ABA levels were elevated in mutants under drought stress- not due to increased biosynthesis, but via the release of conjugated vacuolar ABA-GE through β-glucosidase BG2. Consistent with this, ABA-responsive genes such as RD29A/B and ABF2/3 were more strongly upregulated in mutants than in wild type (WT). Conversely, PP2CA, which encodes a phosphatase involved in ABA negative feedback, was repressed in the absence of GASA3 and AFP1. These results suggest that both genes act as negative regulators of drought tolerance, with AFP1 influencing GASA3 expression.
Altogether, this thesis offers new insights into plant abiotic stress responses and provides a foundation for future functional studies in barley and other crops, potentially guiding breeding strategies for improved stress resilience under changing climates
JAR1-mediated JA-Ile accumulation: a mechanism towards drought stress resistance in <em>Arabidopsis thaliana</em>
Get PDFIn the present work, the regulatory capacity of biologically active jasmonate, jasmonyl-isoleucine (JA-Ile), under normal and progressive drought stress conditions throughout a plant’s life-cycle was investigated. To alter endogenous JA-Ile levels, two different plant lines were used: i) the T-DNA insertion line jar1-11, which contains significantly reduced amount of JA-Ile and ii) complementary to this, a T-DNA insertion line (JAR1-OE) expressing JAR1.1-YFP under the control of the 35S promoter, which results in JAR1 overexpression and enhanced endogenous JA-Ile levels. This line was newly developed within this work. Both lines displayed difference in growth and stress resistance compared to the wild type and each other. Under normal growth conditions jar1-11 plants displayed a larger rosette with narrower leaf blades, while JAR1-OE plants had stunted growth with lateral leaves. And while JAR1-OE was late in flowering, a reciprocal trend was observed in jar1-11. Furthermore, jar1-11 plants were more susceptible to drought stress, while JAR1-OE plants were highly resistant. In line with the difference in JAR1, hormone analysis revealed increased accumulation of JA-Ile in JAR1-OE under drought, while jar1-11 accumulated JA that could not be converted to JA-Ile. In addition, the homeostasis of some precursors and highly abundant catabolic products of JA and JA-Ile were differentially affected in these lines. Global gene expression analysis by RNA-seq revealed a reprogramming of the jasmonate signaling pathway with a positive feedback upregulation in JAR1-OE under drought stress. By contrast, in jar1-11 the biosynthesis of jasmonates was inhibited. Positive feedback in JAR1-OE helps plants to acquire pre-stress tolerance with positive stomatal regulation, anti-oxidant activity and modulation of ABA biosynthesis. This ultimately helps the plants in coping with subsequent drought stress through regulation of the photosynthetic machinery and other biological processes. Furthermore, calmodulin-like protein 12 (CML12) was identified as a potential target of jasmonate signaling. Intriguingly, CML12 behaves differentially at the transcriptional and translational levels to the presence or absence of JAR1 or endogenously added JA-Ile supporting a potential cross-talk between jasmonate and Ca2+-signaling. Finally, the transcription factor AtMYB2 was found to be a regulator of jasmonate signaling as it could control the accumulation of JA and JA-Ile under normal growth as well as drought stress conditions
- …
